Development of Artificial Agonists for a Bacterial Riboswitch

描述（由申请人提供）：抗生素耐药性的出现要求应用新的方法，以有效地对抗许多医学相关的细菌感染。目前使用的有限抗生素需要用新的，严格的和安全的治疗方法来取代，以对抗今天进化的细菌。消灭细菌的一种方法是针对它们最重要的过程之一，新陈代谢。最近发现的RNA结构元件，称为核糖开关，结合细胞代谢物和控制基本代谢基因的表达，为开发人工激动剂以对抗细菌感染提供了独特和独特的靶标。核糖开关存在于信使RNA的非编码区，当代谢物直接与RNA结合时，基因表达被调节。许多核糖开关抑制参与代谢物合成的附近基因的表达，提供了遗传控制的有效反馈机制。一种特殊的核糖开关（glmS核糖开关）与葡萄糖胺-6-磷酸（GlcN 6P）结合，葡萄糖胺-6-磷酸是革兰氏阳性细菌细胞壁的结构单元，并进行自我切割，导致mRNA失活。GlcN 6P的胺功能似乎直接参与RNA催化，而磷酸盐可能在RNA识别配体中发挥作用。为了开发靶向glmS核糖开关的有效人工激动剂/抗生素，对核糖开关-代谢物复合物的结构和功能细节的理解是必不可少的。该基金的目标集中在（1）研究glmS核糖开关中金属离子的结构和催化作用，（2）破译glmS核糖开关的配体识别，以及（3）设计具有刺激glmS核糖开关自切割和控制基因表达能力的非天然激动剂。使用核苷酸类似物干扰作图和抑制（分别为NAIM和NAIS），将确定glmS核糖开关、其配体和金属离子之间的一些长程接触。使用NAIM，glmS核糖开关内的单个化学基团的生物化学贡献将使用修饰感兴趣的原子的核苷酸类似物来定义。使用NAIS，将确定glmS RNA结构内或涉及glmS RNA结构的特定三级氢键伴侣。核糖开关的结构-功能研究将使非天然代谢物样化合物的合理设计成为可能，这些化合物可能作为激动剂/抗生素通过改变基因表达来阻止细菌生长。由于缺乏有效的抗生素，细菌感染的威胁已经成为最重要的问题，因为这些病原体对公众可用的几乎所有抗生素都具有耐药性。对于靶向不同但特异性和必需的代谢途径的新型抗微生物剂的需求是巨大的，例如利用核糖开关来控制基因表达的那些。核糖开关的结构-功能研究将使非天然激动剂的合理设计，最终可以作为抗生素的功能。

DESCRIPTION (provided by applicant): The emergence of antibiotic resistance has required that new approaches be applied in order to effectively fight a host of medically relevant bacterial infections. The limited group of antibiotics, currently in use, need to be replaced with novel, rigorous, and safe treatments in order to combat the evolved bacterium of today. One way to destroy bacteria is to target one of their most essential processes, metabolism. The recent discovery of RNA structural elements, termed riboswitches, that bind cellular metabolites and control expression of essential metabolic genes provides a unique and distinct target for development of artificial agonists to fight bacterial infections. Riboswitches are found in non-coding regions of messenger RNAs, and gene expression is modulated when metabolite binds directly to the RNA. Many riboswitches repress expression of nearby genes involved in the synthesis of the metabolite, providing an efficient feedback mechanism of genetic control. One particular riboswitch (the glmS riboswitch) binds to glucosamine-6-phosphate (GlcN6P), a building block of the cell wall in Gram-positive bacteria, and undergoes self-cleavage resulting in inactivity of the mRNA. The amine functionality of GlcN6P seems to be directly involved in RNA catalysis, whereas the phosphate may play a role in recognition of the ligand by the RNA. In order to develop effective artificial agonists/antibiotics that target the glmS riboswitch, an understanding of the structural and functional details of the riboswitch-metabolite complex is essential. The aims of this grant focus on (1) investigating the structural and catalytic roles of metal ions in the glmS riboswitch, (2) deciphering ligand recognition by the glmS riboswitch, and (3) designing non-natural agonists with the ability to stimulate glmS riboswitch self-cleavage and control gene expression. Using Nucleotide Analog Interference Mapping and Suppression (NAIM and NAIS, respectively) some of the long range contacts between the glmS riboswitch, its ligand, and metal ions will be determined. Using NAIM, the biochemical contribution of a single chemical group within the glmS riboswitch will be defined using nucleotide analogs that modify the atom(s) of interest. Using NAIS, specific tertiary hydrogen bonding partners within or involving the glmS RNA structure will be determined. Structure-function studies of riboswitches will enable rational design of non-natural metabolite-like compounds that might function as agonists/antibiotics to halt bacterial growth through alteration of gene expression. The threat of bacterial infections due to lack of effective antibiotics has come to the forefront as these pathogens become resistant to almost every antibiotic available to the public. The need is great for new classes of anti-microbial agents that target different, but specific and essential, metabolic pathways, such as those which utilize riboswitches to control gene expression. Structure-function studies of riboswitches will enable rational design of non-natural agonists that ultimately could function as antibiotics.